한빛사 논문
Beum-Chang Kang1, Su-Ji Bae1, Seonghyun Lee1, Jeong Sun Lee1, Annie Kim1,2, Hyunji Lee1, Gayoung Baek1, Huiyun Seo1, Jihun Kim1 and Jin-Soo Kim1,*
1Center for Genome Engineering, Institute for Basic Science, Daejeon, Republic of Korea. 2Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
*Corresponding author.
Abstract
Plant organelles including mitochondria and chloroplasts contain their own genomes, which encode many genes essential for respiration and photosynthesis, respectively. Gene editing in plant organelles, an unmet need for plant genetics and biotechnology, has been hampered by the lack of appropriate tools for targeting DNA in these organelles. In this study, we developed a Golden Gate cloning system, composed of 16 expression plasmids (8 for the delivery of the resulting protein to mitochondria and the other 8 for delivery to chloroplasts) and 424 transcription activator-like effector subarray plasmids, to assemble DddA-derived cytosine base editor (DdCBE) plasmids and used the resulting DdCBEs to efficiently promote point mutagenesis in mitochondria and chloroplasts. Our DdCBEs induced base editing in lettuce or rapeseed calli at frequencies of up to 25% (mitochondria) and 38% (chloroplasts). We also showed DNA-free base editing in chloroplasts by delivering DdCBE mRNA to lettuce protoplasts to avoid off-target mutations caused by DdCBE-encoding plasmids. Furthermore, we generated lettuce calli and plantlets with edit frequencies of up to 99%, which were resistant to streptomycin or spectinomycin, by introducing a point mutation in the chloroplast 16S rRNA gene.
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